Omega-3 (n-3) polyunsaturated fatty acids (PUFA), such as alpha-linolenic acid (ALA orC18:3,n-3), eicosapentaenoic acid (EPA or C20:5,n-3), docosapentaenoic acid (DPA or C22:5,n-3) and docosahexaenoic acid (DHA,C22:6,n-3) are major components of cellular membranes with an ever-increasing array of newly discovered physiologic roles such as: maintenance of membrane flexibility, modulation of autoimmunity, inhibition of inflamation and platelet aggregation, reduction of hypertriglyceridemia,inhibition of neoplastic cell proliferation and metastasis. ALA is available from plants, e.g., flax seed and rape seed or canola oils, and some vegetables (e,g,. purslane and spinach). It serves as a catabolic precursor for EPA (1) and DHA (2), which are available for human consumption mainly from marine sources. The ALA to EPA to DHA tamsformation is slow and an in vivo production of 0.3 grams of EPA requires ingestion of about 3.0 grams of ALA. Therefore, marine sources, consisting mainly of oil of cold water fish (menhaden, tuna, salmon, cod, etc.) are used to shortcut the tedious metabolic pathway and provide the organism with the needed PUFA. EQU CH.sub.3 --(CH.sub.2 --CH.dbd.CH).sub.5 --CH.sub.2 --CH.sub.2 --CH.sub.2 --COOH (1) EQU CH.sub.3 --(CH.sub.2 --CH.dbd.CH).sub.6 --CH.sub.2 --CH.sub.2 --COOH (2)
Such cold-water fish oils provide a mixture of omega-3s, containing EPA (up to about 15% of total fatty acids by weight) and DHA (up to about 12% of total fatty acids by weight) were the main source of omega-3 PUFA The refining, deodorization and purification of these oils, or their individual fatty acid components, for human therapeutic use are tedious and expensive processes.
The EPA and DHA in these processed oils are highly oxidation-sensitive if exposed to air. The unpleasant fish odor of the oil returns, even with addition of antioxidants like alpha-tocopherol (vitamin E). DHA is the less oxidation-sensitive of the two PUFA, and its encapsulation under nitrogen ensures a reasonable shelf life. It appears that the more DHA and the less EPA are present in an oil, the more stable the oil becomes and its shelf life is prolonged.
Recently, two new, natural sources of DHA-rich oils, with a preponderance of DHA-containing mixed triglycerides, have been identified and developed: 1) oil from the ocular orbit fatty tissue of tuna, which provides an odorless source of at least 27% DHA of total fatty acids by weight and only about 7-8% EPA. This oil, sold in the United States under the trade name DHA Maguro, requires only minimum processing under nitrogen, at low temperatures, and. 2) oils from algae and algae-like microorganisms that provide odorless oils that contain up to 50% DHA, are free of EPA or contain only small amounts of EPA and essentially no other polyunsaturated fatty acids (W. R. Barclay, K. M. Meager, and J. R. Abril, "Heterotrophic production of long chain omega-3 fatty acids utilizing algae and algae-like microorganisms," J. Appl. Phycology, 1994, 6, 123-129). The pharmaceutical utility of these oils can be enhanced through control of the biotechnologic fermentation process and its degree of sterility. These oils can be further enriched with docosahexaenoylethylester and/or various docosahexaenoyl-containing phospholipids such as: 1-stearoyl, 2-docosahexaenoyl phosphatidylcholine or 1-stearoyl, 2-docosahexaenoyl phosphatidyl ethanolamine.
The present invention is building on the availability of these odorless, easy to sterilize high-DHA content triglyceride-based oils for the theraeutic applications described herewithin.
The understanding of the role of PUFA, particularly EPA and DHA and their esters with alkanols and glycerol in therapeutic applications related to cancer such as prevention of cancers, inhibition of tumor growth and prevention of dissemination of cancer cells (metastases) is steadily increasing Spector, A. A., and Bums, C. P., "Biological and therapeutic potential of membrane lipid modification in tumors," Cancer Res., 1987, 47, 4529-4537; Rose, D. P. and Cohen, L. A., "Effects of dietary menhaden oil and retinyl acetate on the growth of DU 145 human prostatic adenocarcinoma cells transplanted into athymic nude mice," Carcinogenesis, 1998, vol.9, no.4, 603-605; Bums, CP., and Spector, A. A, "Effects of lipids on cancer therapy," Nutr. Rev., 1990, vol. 48, no.6, 233-240; Lowell, J. A., Pames, H. L., and Blackburn, G. L., "Dietary immunomodulation: beneficial effects on oncogenesis and tumor growth," Crit. Care Med., 1990, 18(2 Suppl): S145-8; Grunfeld, C., and Feingold, K. R., "Tumor necrosis factor, interleukin, and interferon induced changes in lipid metabolism as part of host defense," Proc. Soc. Exp. Biol. Med., 1992, 200(2): 224-7; De Vries, C. E., and van Noorden, C.,J., "Effects of dietary fatty acid composition on tumor growth and metastasis," Anticancer Res., 1992, 12(5):1513-22; Isffan, N. W., Wan, J. M., and Bistrian, B. R., "Nutrition and tumor promotion: in vivo methods for measurement of cellular proliferation and protein metabolism," J. Parenter. Enteral. Nutr., 1992, 16(6 Suppl): 76-82S; Kinsella, J. E., and Black, J. M., "Effects of polyunsaturated fatty acids on the efficacy of antineoplastic agents toward L5178Y lymphoma cells," Biochem. Pharm., vol. 45, no. 9, 1881-1887; Connolly, J. M., and Rose, D. P., "Effects of fatty acids on invasion through reconstituted basement membrane (`Matrigel`) by a human breast cancer cell line," Cancer Letters 1993, 75, 137-142; Gonzalez, M. J., et al., "Dietary fish oil inhibits human breast carcinoma growth: a function of increased lipid peroxidation," Lipids, vol.28, no.9, 827-832; Rose, D. P., and Connolly, J. M., "Effects of dietary omega-3 fatty acids on human breast cancer growth and metastases in nude mice," J. Nat. Cancer Inst. 1993, 85 (21), 1743-47!.
Very recent data from a study aimed at comparing three concentrated n-3 PUFA preparations-ethyl esters, free fatty acids and re-esterified triglycerides-with placebo oil in a double-blinded study showed that the bioavailability of orally administered EPA and DHA, from highly concentrated preparations containing EPA and DHA as re-esterified triglycerides, was significantly better than ethyl esters and even better than free acids. (Kilde: Dyerberg J. "Bioavailability of n-3 fatty acids formulations". European Society for Clinical Investigation, Annual Meeting April, 1995. Workshop: Preventive Strategies in Vascular Disease: Focus on n-3 Fatty Acids). Additional data indicates that specific triacyl glycerols with EPA and DHA in the 2-(sn2) position of glycerol, provides a readily absorbed source of long chain omega-3 PUFA for nutritional supplementation purposes. (Christensen, M. S., et al, "Intestinal absorption and limphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids: dependence on intramolecular triacylglycerol structures", Am. J. Clin. Nutr. 1995, 61, 56-61.)
In prior art. DHA was tested to determine its mode of action as an anticancer agent. It is shown that DHA, either as the free fatty acid or as 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine, is incorporated in tumor plasma membranes (e.g. T27A leukemia cell membranes) and makes them substantially more permeable, explaning at least in part its anti-tumor properties. For details, see Stillwell W., Ehringer W. and Jensid L. J., "Docosahexaenoic acid increases permeability of lipid vesicles and tumor cells", Lipids 1993, 28(2): 103-8; Jenski L. J., Sturdevant L. K., Ehringer, W. D., and Stillwell W., "Omega-3 fatty acid modification of membrane structure and function. I. Dietary manipulation of tumor call susceptibility to cell- and complement-mediated lysis", Nutr. Cancer 1993, 19(2): 135-46; Pascale A. W., Ehringer, W. D., Stillwell W., Sturdevant L. K., and Jenski L. J., "Omega3 fatty acid modification of membrane structure and function. II. Alteration by docosahexaenoic acid of tumor cell sensitivity to immune cytolysis", Nutr. Cancer 1993, 19(2): 147-57!.
Docosahexaenoic acid has an inhibiting effect on tumor growth. Lipid peroxidation appears to be an important factor in the inhibition of tumor growth. Both EPA and DHA seem to autooxidize enzymatically by cytochrome P450, cyclooxygenase and lipoxygenase. For example, DHA in 1-oleoyl 2-docosahexaenoyl-sn glycero3-phosphocholine (1-oleoyl 2-DHA-PC) was found to be a potent inhibitor of 5-lipoxygenase Matsumoto K, Morita I., Hibino H. and Murota S., "Inhibitory effect of docosahexaenoic acid-containing phospholipids on 5 lipcxygenase in rat basophilic leukemia cells", Prostaglandins Leukot Essent Fatty Acids 1993, 49(5): 861-6!.
In vitro studies demonstrated that docosahexaenoic acid is cytotoxic at concentrations greater than or equal to 20 microM after 48-72 h in culture. The extension of these results to situations in vivo could lead to use of docosahexaenoic acid for delaying leukemia progression or in adjuvant chemotherapy Anel A., Naval J., Desportes P., Gonzalez B., Uriel J., and Pineiro A., "Increased cytotoxicity of polyunsaturated fatty acids on human tumoral B and T-cell lines compared with normal lymphocytes", Leukemia, 1992, 6(7): 680-8!.
Also, omega-3 fatty acids (mainly DHA) inhibited MDA-MB-435 human breast cancer cell line invasion in vitro as demonstrated by Connolly J. M. and Rose D. P., "Effects of fatty acids on invasion through reconstituted basement membrane (`Matrigel`) by a human breast cancer cell line", Cancer Lett, 1993, 75(2): 137-42!.
The roles of DHA in inhibition of growth and metastasis of murine transplantable mammary tumor have been recently described Kinoshita K., Noguchi M., Earashi M., Tanaka M. and Sasaki T., "Inhibitory effects of purified eicosapentaenoic acid and docosahexaenoic acid on growth and metastasis of murine transplantable mammary tumor", In vivo, 1994, 8 (3): 371-4!.
The growth and sensitivity of neoplastic cells to chemotherapeutic agents may be altered by the type of fatty acids incorporated in cell membranes. For example, a prior enrichment of cellular components with DHA enhances the toxic action of anticancer drugs Kinsella J. E., and Mark Black J., "Effects of polyunsaturated fatty acids on the efficacy of antineoplastic agents toward L5178Y lymphoma cells", Biochem. Pharmacology, 1993, 45(9), 1881-1887; Wagner B. A., Buettner G. R., and Burns C. P., "Increased generation of lipid-derived and asoorbate free radicals by L1210 cells exposed to the ether lipid edelfosine", Cancer Res., 1993, 53: 711-713; Buettner G. R., Kelley E. E., and Burns, C. P., "Membrane lipid free radicals produced from L1210 murine leukemia cells by photofrin photosensitization: An electron paramagnetic resonance spin trapping study", Cancer Res., 1993, 53:3670-3673; Petersen E. S., Kelley E. E., Modest E. J., and Burns C. P., "Membrane lipid modification and sensitivity of leukemic cells to the thioether lipid analogue BM 41.440", Cancer Res., 1992, 52: 6263-6269!.
A recent series of studies indicated that DHA containing diacyl- and alkenylacylglycerophosphoethanolamine (DHA-diacylGPE, and DHA-alkenyl acyGPE) are not susceptible to deacylation and release of the DHA by the cytosolic phospholipase A2 (cPLA2). This behaviour was in stark contrast with the behavior of arachidonic acid- and EPA-containing diacylGPE and alkenylacyGPE derivatives. The authors assume that clear discrimination of DHA from EPA and archidonic acid by cPLA2, may provide a new basis for understanding the beneficial effects of DHA. Shikano, M., Masuzawa Y., Yazawa K., Takayama K., Kudo I., and Inoue K., "Complete discrimination of docosahexaenoate from arachidonate by 85 kDa cytosolic phospholipase A.sub.2 during the hydrolysis of diacyl- and alkenylacylglycerophosphoethanolamine", Biochin Biophys. Acta, 1994, 1212:211-216!.
Another study found that DHA is incorporated more readily into malignant brain tumour tissue (e.g. glial tumours, astrocytomas and glioblastomas) than into normal brain tissue due primarily to an increased utilization of fatty acids by tumor cells Nariai T., Greig N. H., DeGeorge J. J., Genka S. and Rapoport S. I., "Intravenously injected radiolabelled fatty acids image brain tumour phospholipids in vivo: differential uptakes of palmitate, arachidonate and docosahexaenoate", Clin. Exp. Metastasis, 1993, 11:141-149!.
Recently, is shown that DHA suppresses the formation and growth of colon cancer and has a preventive effect on colon carcinogenesis Takahashi M., Minamoto T., Yamashita N., Yazawa K, Sugimura T., and Esumi H., "Reduction in formation and growth of 1,2-dimethylhydrazine-induced aberrant crypt foci in rat colon by docosahexaenoic acid", Cancer Res., 1993, 53 (12): 2786-9; Anti M., Marra G., Armelao, F., Bartoli G. M., Ficarelli R., Percesepe, A, De Vitis I., Maria G., Sofo L., Rapaccini G. L., et al., "Effect of omega-3 fatty acids on rectal mucosal cael proliferation in subjects at risk for colon cancer", Gastroenterology 1992, 103(3), 883-91; Gastroenterology 1993, 104(4):1239-41!.
It was recently emphasised that the intestinal absorption and lymphatic transport of DHA depends on intramolecular triacylglycerol structure. Specific triacylglycerols with DHA in the sn-2 position and medium-chain saturated acyl substituents in the sn-1 and sn-3 positions of are of value in delivering DHA and in ensuring adequate bioavailability following enteral administration. Interesterified or re-esterified triacylglycerols open new possibilities for designing special lipids for particular therapeutic purposes. Christensen M. S., Hoy C. E., Becker C. C. and Redgrave T. G., "Intestinal absorption and lymphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids: dependence on intramolecular triacylglycerol structure", Am. J. Clin. Nutr., 1995, 61: 56-61; Specific and random triglicerides are described in U.S. Pat. Nos. 5,227,403, July, 1993 Seto et al.: Fats and oils having superior digestibility and absorptivity used as nutrients; and 4,701,469 October, 1987 Mendy et al., Preparation, dietetic applications and compositions of specific triglycerides.!
Another recent study of arachidonic acid metabolism in benign and malignant prostatic tissue, related the presense of high levels of prostaglandin E2 (PGE2) in the malignant prostatic tissue (as opposed to cells from benign prostatic hyperplasia patients, and normal subjects). Since arachidonic acid is responsible for PGE2 formation as well as diacyl glycerol (DAG) formation in the neoplastic malignant cells and omega-3 PUFAs from fish oil significantly inhibited PGE2 and DAG formation in the neoplastic cells, it is reasonable to assume that either EPA, or DHA or both present in the fish oil exhibit possible therapeutic effectc in human prostate cancer.
Significant increase in survival of T27A murine leukemia cell-containing, tumor-bearing mice was reported when the mice were treated intraperitoneally with small unilamellar vesicle preparations of 1-stearoyl, 2-docosahexaenoyl phosphatidylcholine. Although a positive effect was observed also with the derivative in which alpha-linolenic acid replaced docosahexaenoic acid, the strongest survival effect was that of the DHA derivative. (Jenski, L. J. et al., "Antitumor effects of omega-3 fatty acid-containing lipid vesicles administerd in situ". Abstract #134, 2nd International Congress of the ISSFAL International Society for the Study of Fatty Acids and Lipids, Jun. 7-10, 1995, NIH Bethesda Md., on "Fatty Acids and Lipids From Cell Biology To Human Disease"). The prior art is silent regarding the use of DHA rich triglyceridic oils in treating neoplastic diseases.